1. Field of the Invention
The present invention pertains to genetic screening methods, related cells and culturing media thereof, useful in identifying clones of mammalian cells expressing the polypeptide of interest. The methods allows for high throughput screening of recombinant cells for elevated levels of expression of polypeptide of interest. The present invention also provides a screening method useful in screening and isolating clones of mammalian cells expressing high levels of immunoglobulin.
2. Related Background
Recombinant proteins (r-proteins) are an emerging class of therapeutic agents. To obtain a stable clone for recombinant protein production usually requires the transfection of cells with an expression vector containing gene of interest and a dominant genetic marker. Typically, for the selection of stable transfectants, a selectable marker such as an antibiotic resistance gene is transfected along with the target gene of interest. Selection is then carried out in the presence of the specific antibiotic. Cells that have taken up the expression vector DNA survive in appropriate selection media.
Currently, cloning of stably transfected cells relies on performing a series of limiting dilution procedures, a time consuming and labor-intensive process. For example, many commonly used mammalian expression systems are based on stably transfected Chinese Hamster Ovary (CHO) cells and transfection efficiencies in this system range from 10-60% of cells taking up the vector DNA. However, a wide variation in recombinant gene expression exists among clones that stably incorporate the foreign DNA into the genome due to the position effect by which different regions of the chromosome modulate the expression of the transfected gene. Many hundreds, even thousands of transfected clones are typically screened for random high producers because of the random variation in recombinant protein production. Therefore in many cases, screening for high producers has been one of the rate limiting procedures in developing of cell lines expressing r-proteins due to the huge amount of cells to screen and the complicated assays to perform.
Soluble proteins interact with their corresponding antibody to form a precipitate in solid or semisolid substrates such as agarose. One such application is the immunoplate assay used to detect mouse myeloma mutants. Briefly, cells are cloned in soft agarose over feeder layers that undergo contact inhibition. Antibody or antigen reactive with the immunoglobulin that is secreted by the cloned cells is added to the plate and diffuses through the agarose forming an antigen-antibody precipitate surrounding the clone. This precipitate appears as a collection of dark granules and specks under low or medium power with an inverted microscope. This assay was used not only to look for mutants of hybridoma and myeloma cells, but also to clone hybridomas and identify subclones producing the desired antibody. It can also be used to identify high producers.
However, several difficulties were reported previously when using this semi-solid agarose technique for screening clones producing the desired antibody. For example, poor growth of mammalian cells is caused by inability to utilize the correct temperature to seed cells while agarose is cooling. Another common problem is the difficulty in viewing the precipitate in the agarose media even under a microscope. It is also difficult to correlate the precipitate size to the level of protein secretion.
Accordingly, there is a need to provide improved and/or modified screening methods, which overcome and/or substantially ameliorate one or more of these and other problems known in the art.